Project Summary While axis specification during embryogenesis has been studied extensively in a variety of animal models, much less is known about how body axes are modified and repatterned during regeneration and asexual reproduction. Failure to re-establish proper axial polarity after amputation can inhibit regeneration, though experimental restoration of proper polarity signals has been shown to rescue failed regeneration programs. These results suggest that proper respecification of axis polarity is vital to subsequent postembryonic development. This proposal aims to identify developmental mechanisms that allow for the alteration and re-specification of axial polarity within adult body plans. Acoel worms have emerged as an innovative and novel model system uniquely suited to study postembryonic axis modification with species undergoing anterior-posterior (AP) axis reversal during budding and splitting of the midline and left-right (LR) axis polarity as part of longitudinal fission. Aim 1 of this proposal seeks to test hypotheses based on preliminary data that Hedgehog signaling plays a novel role in the loss of axial polarity at the site of bud initiation and subsequent Wnt signals are modulated to re-establish a reversed AP axis in budding tissues. Aim 1 also characterizes Notch and Slit signaling as key molecular signals involved in the loss of midline identity and re-establishment of LR axis polarity during longitudinal fission. Knockdown of genes encoding ligands, receptors, and effectors will elucidate the specific function of candidate signaling pathways during all stages of polarity loss and modification. Aim 2 takes a more unbiased approach and utilizes deep sequencing technology to characterize alterations in gene expression during the initiation and progression of axial respecification. Comparing the transcriptomes of normal tissues to tissues in polarity transition regions will identify other genes that modulate the loss and eventual respecification of axis polarity. In addition, comparing gene expression profiles of wild-type tissues to those experimentally manipulated by RNAi of candidate genes tested in Aim 1 will identify downstream transcripts that participate in polarity modification and re-establishment. Differentially expressed gene candidates will be characterized functionally to identify molecular signals that disrupt established body axes, confer developmental plasticity, and allow novel body axes to form. Identified candidate genes and signaling mechanisms that allow for in vivo axis respecification offer valuable insights into possible means by which to reprogram damaged tissues in regeneration- deficient organisms. This work will provide a solid foundation for subsequent investigations that may ultimately lead to regeneration of damaged human tissues or limbs.